This goal of this project is to better understand the connection between aging and osteoarthritis (OA). OA is a painful disease of the joints that affects an estimated 27 million people in the United States. While it is well established that the risk for OA increases with age, the reason for this is not fully known. One potential explanation is that chondrocytes, the cells that make up cartilage tissue, exhibit a particular form of dysfunction associated with aging known as cellular senescence. Senescent cells are characterized by the inability to divide, the excessive production of inflammatory molecules, and the expression of a gene known as p16Ink4a. This project will explore the hypothesis that cellular senescence increases in joints during the development or OA associated with aging or injury, and that chondrocyte senescence functionally contributes to OA development. The scientific approach is to utilize mice that have been genetically engineered to have distinct functions in place of the normal expression of p16Ink4a. The first aim will track the development of senescence at the single cell level by analyzing mice that express the fluorescent protein tdTomato instead of p16Ink4a. This will allow precise identification of the frequency of senescence during the development of OA with aging and after joint injury. Furthermore, cells will be sorted with flow cytometry based on tdTomato expression and the transcriptional profile of senescent cells will be compared to non-senescent cells using RNA-sequencing. The second aim will assess the progression of OA in mice that have a chondrocyte-specific deletion of p16Ink4a upon administration of tamoxifen in adulthood. Because these mice are expected to exhibit less chondrocyte senescence, observations that OA is reduced in the context of aging or joint injury would support the hypothesis that maintaining a senescent state is functionally important to the development of OA. The long-term objective of this work is to identify new strategies for preventing or slowing the progression of OA, including the clearance of senescent cells from the joint. This fellowship grant will also train the principle investigator in technique of murine genetics and cartilage aging in order to establish a research career in the field.